The present invention relates generally to the field of recombinant DNA technology, to means and methods utilizing such technology in the discovery of a broad class of non-human animal interferons and to the production thereof and to the various products of such production and their uses.
More particularly, the present invention relates to the isolation and identification of DNA sequences encoding non-human animal interferons and to the construction of recombinant DNA expression vehicles containing such DNA sequences operably linked to expression-effecting promoter sequences and to the expression vehicles so constructed. In another aspect, the present invention relates to host culture systems, such as various microorganism and vertebrate cell cultures transformed with such expression vehicles and thus directed in the expression of the DNA sequences referred to above. In yet other aspects, this invention relates to the means and methods of converting the novel end products of such expression to entities, such as pharmaceutical compositions, useful for the prophylactic or therapeutic treatment of non-human animals. In addition, this invention relates to various processes useful for producing said DNA sequences, expression vehicles, host culture systems and end products and entities thereof and to specific and associated embodiments thereof.
The present invention arises in part from the discovery of the DNA sequence and deduced amino acid sequence encoding a series of bovine alpha interferons, including the 3xe2x80x2- and 5xe2x80x2-flanking sequences thereof facilitating their in vitro linkage into expression vehicles. These, in turn, enable the development of the means and methods for producing, via recombinant DNA technology, sufficient amounts of non-human animal interferons, so as to enable, in turn, the determination of their biochemical properties and bioactivity, making possible their efficient production for commercial/biological exploitation.
The publications and other materials hereof used to illuminate the background of the invention, and in particular cases, to provide additional details respecting its practice are hereby incorporated herein by this reference, and for convenience, are numerically referenced by the following text and respectively grouped in the appended bibliography.
A. Non-human Animal Interferons
Interferon components have been isolated from tissues of various phylogenetic species lower than human (1,2,3). Activity studies conducted with these interferons have demonstrated varying degrees of antiviral effects in the requisite host animal (3,4,5,6). It also has been demonstrated that these interferons are not always species specific. For example, preparations of bovine interferons isolated from tissues, had antiviral activity on monkey and human cells (7). Likewise, human interferons have been found active in various cells of phylogenetically lower species (see 7).
This species interactivity is doubtless due to a high degree of homologous conservation, both in amino acid composition and sequence, amongst the interferons. However, until now, this explanation remained theoretical because the amounts and purities of non-human animal interferons that have been obtainable were insufficient to carry out unambiguous experiments on the characterization and biological properties of the purified components versus several of their human counterparts (8,9,10,11,12).
In any event, despite these low amounts and purities, a causal connection between interferon and anti-viral activity in the requisite animal host has been established. Thus, the production of non-human animal interferons in high yields and purities would be very desirable in order to initiate and successfully conduct animal bioassay experiments leading toward commercial exploitation in the treatment of animals for viral infections and malignant and immunosuppressed or immunodeficient conditions. In addition, the production of isolated non-human animal interferon species would enable their characterization, both physical and bioactive, and thus provide a basis for categorization and consequential comparative studies with counterpart human interferon species (see 8 to 20).
The studies done with non-human animal interferons, until the present invention, being restricted to the use of rather crude preparations, due to their very low availability, nevertheless suggest very important biological functions. Not only have the class of non-human animal interferons a potent associated therapeutic antiviral activity, but also potential as a prophylactic adjunct with vaccine and/or antibiotic treatment, clearly pointing to very promising clinical and commercial candidates.
It was perceived that the application of recombinant DNA technology would be a most effective way of providing the requisite larger quantities of non-human animal interferons necessary to achieve clinical and commercial exploitation. Whether or not the materials so produced would include glycosylation which is considered characteristic of native derived material, they would probably exhibit bioactivity admitting of their use clinically in the treatment of a wide range of viral, neoplastic, and immunosuppressed conditions or diseases in non-human animals.
B. Recombinant DNA Technology
Recombinant DNA technology has reached the age of some sophistication. Molecular biologists are able to recombine various DNA sequences with some facility, creating new DNA entities capable of producing copious amounts of exogenous protein product in transformed microbes. The general means and methods are in hand for the in vitro ligation of various blunt ended or xe2x80x9cstickyxe2x80x9d ended fragments of DNA, producing potent expression vehicles useful in transforming particular organisms, thus directing their efficient synthesis of desired exogenous product. However, on an individual product basis, the pathway remains somewhat tortuous and the science has not advanced to a stage where regular predictions of success can be made. Indeed, those who portend successful results without the underlying experimental basis, do so with considerable risk of inoperability.
The plasmid, an extrachromosomal loop of double-stranded DNA found in bacteria and other microbes, often times in multiple copies per cell, remains a basic element of recombinant DNA technology. Included in the information encoded in the plasmid DNA is that required to reproduce the. plasmid in daughter cells (i.e., an origin of replication) and ordinarily, one or more phenotypic selection characteristics such as, in the case of, bacteria, resistance to antibiotics, which permit clones of the host cell containing the plasmid of interest to be recognized and preferentially grown in selective media. The utility of plasmids lies in the fact that they can be specifically cleaved by one or another restriction endonuclease or xe2x80x9crestriction enzymexe2x80x9d, each of which recognizes a different site on the plasmid DNA. Thereafter heterologous genes or gene fragments may be inserted into the plasmid by endwise joining at the cleavage site or at reconstructed ends adjacent to the cleavage site. Thus formed are so-called replicable expression vehicles. DNA recombination is performed outside the cell, but the resulting xe2x80x9crecombinantxe2x80x9d replicable expression vehicle, or plasmid, can be introduced into cells by a process known as transformation and large quantities of the recombinant vehicle obtained by growing the transformant. Moreover, where the gene is properly inserted with reference to portions of the plasmid which govern the transcription and translation of the encoded DNA message, the resulting expression vehicle can be used to actually produce the polypeptide sequence for which the inserted gene codes, a process referred to as expression.
Expression is initiated in a region known as the promoter which is recognized by and bound by RNA polymerase. In the transcription phase of expression, the DNA unwinds, exposing it as a template for initiated synthesis of messenger RNA from the DNA sequence. The messenger RNA is, in turn, translated into a polypeptide having the amino acid sequence encoded by the mRNA. Each amino acid is encoded by a nucleotide triplet or xe2x80x9ccodonxe2x80x9d which collectively make up the xe2x80x9cstructural genexe2x80x9d, i.e. that part which encodes the amino acid sequence of the expressed polypeptide product. Translation is initiated at a xe2x80x9cstartxe2x80x9d signal (ordinarily ATG, which in the resulting messenger RNA becomes AUG). So-called stop codons define the end of translation and, hence, of production of further amino acid units. The resulting product may be obtained by lysing, if necessary, the host cell, in microbial systems, and recovering the product by appropriate purification from other proteins.
In practice, the use of recombinant DNA technology can express entirely heterologous polypeptidesxe2x80x94so-called direct expressionxe2x80x94or alternatively may express a heterologous polypeptide fused to a portion of the amino acid sequence of a homologous polypeptide. In the latter cases, the intended bioactive product is sometimes rendered bioinactive within the fused, homologous/heterologous polypeptide until it is cleaved in an extracellular environment (21, 22).
C. Cell Culture Technology
The art of cell or tissue cultures for studying genetics and cell physiology is well established. Means and methods are in hand for maintaining permanent cell lines, prepared by successive serial transfers from isolate normal cells. For use in research, such cell lines are maintained on a solid support in liquid medium, or by growth in suspension containing support nutriments. Scale-up for large preparations seems to pose only mechanical problems (See generally 23,24).
The present invention is based upon the discovery that recombinant DNA technology can be used to successfully produce non-human animal interferons, and each of them, preferably in direct form, and in amounts sufficient to initiate and conduct biological testing as prerequisites to market approval. The product is suitable for use, in all of its forms, in the prophylactic or therapeutic treatment of non-human animals, notably for viral infections and malignant and immunosuppressed or immunodeficient conditions. Its forms include various possible oligomeric forms which may include associated glycosylation as well as allelic or other, induced (such as via site directed mutagenesis of the underlying DNA) variations of individual members or family units. The products are produced by genetically engineered microorganisms or cell culture systems. Thus, the potential now exists to prepare and isolate non-human animal interferons in a more efficient manner than has been possible. One significant factor of the present invention, in its most preferred embodiment, is. the accomplishment of genetically directing a microorganism or cell culture to produce a representative non-human animal interferon, bovine interferon, in isolatable amounts, produced by the host cell in mature form.
The present invention comprises the non-human animal interferons thus produced and the means and methods of their production. The present invention is further directed to replicable DNA expression vehicles harboring gene sequences encoding non-human animal interferons in expressible form. Further, the present invention is directed to microorganism strains or cell cultures transformed with the expression vehicles described above and to fermentation media comprising such transformed strains or cultures, capable of producing non-human animal interferons.
In still further aspects, the present invention is directed to various processes useful for preparing said interferon gene sequences, DNA expression vehicles, microorganism strains and cell cultures and to specific embodiments thereof. Still further, this invention is directed to the preparation of the fermentation media of said microorganisms and cell cultures. Further, in certain host systems, vectors can be devised to produce the desired non-human animal interferon, secreted from the host cell in mature form. The interferon containing the signal sequence derived from the 5xe2x80x2-flanking region of the gene proper is believed to be transported to the cellular wall of the host organisms where, aiding in such transport, the signal portion is cleaved during the secretion process of the mature interferon product. This embodiment enables the isolation and purification of the intended mature interferon without resort to involved procedures designed to eliminate contaminants of intracellular host protein or cellular debris.
In addition, this invention is specifically directed to the preparation of a bovine interferon representative of the class of non-human animal interferons embraced herein, produced by direct expression in mature form.
Reference herein to the expression xe2x80x9cmature non-human animal interferonxe2x80x9d connotes the microbial or cell culture production of non-human animal interferon unaccompanied by the signal peptide or presequence peptide that immediately attends translation of the non-human animal interferon mRNA. Mature non-human animal interferon, according to the present invention, is thus provided, having methionine as its first amino acid (present by virtue of the ATG start signal codon insertion in front of the structural gene) or, where the methionine is intra- or extracellularly cleaved, having its normally first amino acid. Mature non-human animal interferon can also be produced, in accordance herewith, together. with a conjugated protein other than the conventional signal polypeptide, the conjugate being specifically cleavable in an intra- or extracellular environment (see 21). Finally, the mature non-human animal interferon can be produced by direct expression without the necessity of cleaving away any extraneous, superfluous polypeptide. This is particularly important where a given host may not, or not efficiently, remove a signal peptide where the expression vehicle is designed to express the mature interferon together with its signal peptide. The thus produced mature interferon is recovered and purified to a level fitting it for use in the treatment of viral, malignant, and immunosuppressed or immunodeficient conditions.
Non-human animal interferons hereof are those otherwise endogenous to the animal organism including, in nomenclature analogous to human interferons, animal alpha (leukocyte), beta (fibroblast) and gamma (immune) interferons. All three series have been identified in an animal model. Further, based upon the bovine example, the non-human animal alpha series is composed of a family of proteins as in the human case; those investigated have a lower degree of homology to the corresponding human alpha interferons than either those non-human animal interferons have amongst themselves or the human alpha interferons have amongst themselves. In addition, the bovine beta series is composed of a family of proteins, distinct from the human case. In addition, this invention provides interspecies and intrafamily hybrid interferons, by taking advantage of common restriction sites within the genes of the various non-human animal interferons hereof and recombining corresponding portions, according to known methods (see 57).
In any event, the non-human animal interferons embraced by this invention include those normally endogenous to animals of the avian, bovine, canine, equine, feline, porcine, ovine, piscine, and porcine families. In particular, the present invention provides interferons of cloven-hoofed animals such as cattle, sheep and goats. The interferons provided by this invention find application as antiviral and antitumor agents in the respective host animal. For example, bovine interferons would find practical applications in treating respiratory complex in cattle, either in conjunction with (per se known) antibiotics as a therapeutic component or with vaccines as a prophylactic component. Class utility, demonstrated as described above, would extend to other bovine, and to goats, sheep, pigs, horses, dogs, cats, birds and fish. In horses, dogs, cats and birds, the antitumor effect of the corresponding interferons could be expected to be especially important commercially.
Thus, for applications to particular host non-human animals, advantage can be taken in accordance herewith of demonstrated or otherwise manifest interspecies activity such that, for a given example, a given porcine interferon could find useful application in treating a bovine host. This may be particularly useful where, given a particular recombinant system or host according to the general enablement hereof, a particular interferon may be particularly susceptible to commercial exploitation whilst retaining essential bioactivity against a range of conditions not necessarily specific to the same family as that to which the given interferon belongs phylogenetically. In all events, such interspecies utility as can be determined according to analogous testing protocols is within the ambit of the present invention. See, for example, Ohman et al., Antiviral Research 7, 187 (1987) and the references cited therein, which are hereby incorporated by reference.
The following rationale, described with reference to bovine interferon as a representative of the class, may be employed for obtaining various non-human animal interferons hereof, in accordance with this invention:
1. Bovine tissues, for example bovine pancreas tissue, were reduced to frozen powder and treated to digest RNA and protein materials and provide, on precipitation, high molecular weight bovine DNA.
2. The high molecular weight DNA was partially digested for random cutting with respect to gene locus.
3. The resultant DNA fragments were size-fractionated giving from 15 to 20 kilo base pair fragments.
4. The resultant fragments of Step 3 were cloned using a xcex Charon 30 phage vector.
5. The thus prepared vectors were packaged in vitro to infectious phage particles containing rDNA to provide a phage library. This was amplified by propagation on bacterial cells to about 106 fold. The phage were plated to virtual confluence on a lawn of bacteria and screened for hybridization with a radioactive human interferon probe.
6. From the appropriate clones the corresponding DNA was isolated and restriction mapped and analyzed by Southern hybridization. Restriction fragments containing bovine interferon genes were subcloned into plasmid vehicles and then sequenced.
7. The sequenced DNA was then tailored in vitro for insertion into an appropriate expression vehicle which was used to transform an appropriate host cell which was, in turn, permitted to grow in a culture and to express the desired bovine interferon product.
8. Bovine interferon thus produced has 166 amino acids in its mature form, beginning with cysteine, and 23 in the presequence, and is very hydrophobic in character. Its monomeric molecular weight has been calculated at about 21,409. It displays characteristics similar to human leukocyte interferons (8,9,10,11) and has been found to be about 60 percent homologous to a human leukocyte interferon.
Having isolated and identified the DNA encoding a particular non-human animal interferon in accordance herewith, it falls within the skill of the art as generally described and referenced herein to utilize that DNA as a sequence, or subsequence, useful to probe for other non-human interferons, of the same or different families, by application using the appropriate genomic library or other appropriate DNA source. Alternatively, synthetic probes of various lengths can be prepared based upon the sequence identified, or a genetically degenerate encoding form thereof, or the entire DNA sequence, could be Synthesized according to contemporary skill in the art.
Having thus provided such DNA sequences, it similarly falls within the skill in the art, as generally described and referenced herein, to configure it in a number of equivalent expression systems for use with appropriate recombinant hosts.
Specific embodiments for such manipulations as described herein lay foundation, along with the extant art, for producing non-human animal interferons via a variety of enabled recombinant expression systems and hostsxe2x80x94vide supra. Likewise, it belongs to those skilled in the art of animal husbandry to test the thus produced interferons for bioactivity in members of the phylogenetically identical or equivalent families of non-human animals, the results of such bioactivity testing in turn confirming any interspecies and/or interfamilial utility.
A. Microorganisms/Cell Cultures
1. Bacterial Strains/Promoters
The work described herein was performed employing, inter alia the microorganism E. coli K-12 strain 294 (end A, thixe2x88x92, hsrxe2x88x92, khsm+) (25). This strain has been deposited with the American Type Culture Collection, ATCC Accession No. 31446. However, various other microbial strains are useful, including known E. coli strains such as E. coli B, E. coli X 1776 (ATCC No. 31537) and E. coli W 3110 (Fxe2x88x92, xcexxe2x88x92, protrophic) (ATCC No. 27325), E. coli DP 50 SuPF (ATCC No. 39061, deposited Mar. 5, 1982), E. coli JM83 (ATCC No. 39062, deposited Mar. 5, 1982) or other microbial strains many of which are deposited and (potentially) available from recognized microorganism depository institutions, such as the American Type Culture Collection (ATCC)xe2x80x94cf. the ATCC catalogue listing (See also 26, 26a). These other microorganisms include, for example, Bacilli such as Bacillus subtilis and other enterobacteriaceae among which can be mentioned as examples Salmonella typhimurium and Serratia marcesans, utilizing plasmids that can replicate and express heterologous gene sequences therein.
As examples, the beta lactamase and lactose promoter systems have been advantageously used to initiate and sustain microbial production of heterologous polypeptides. Details relating to the make-up and construction of these promoter systems can be obtained by reference to (27) and (28). More recently, a system based upon the tryptophan operon, the so-called trp promoter system, has been developed. Details relating to the make-up and construction of this system have been published by Goeddel et al. (12) and Kleid et al. (29). Numerous other microbial promoters have been discovered and utilized and details concerning their nucleotide sequences, enabling a skilled worker to ligate them functionally within plasmid vectors, have been publishedxe2x80x94see (30).
2. Yeast Strains/Yeast Promoters
The expression system hereof may also employ the plasmid YRp7 (31, 32, 33), which is capable of selection and replication in both E. coli and the yeast, Saccharomyces cerevisiae. For selection in yeast the plasmid contains the TRP1 gene (31, 32, 33) which complements (allows for growth in the absence of tryptophan) yeast containing mutations in this gene found on chromosome IV of yeast (34). One useful strain is strain RH218 (35) deposited at the American Type Culture Collection without restriction (ATCC No. 44076). However, it will be understood that any Saccharomyces cerevisiae strain containing a mutation which makes the cell trp1 should be an effective environment for expression of the plasmid containing the expression system. An example of another strain which could be used is pep4-1 (6). This tryptophan auxotroph strain also has a point mutation in TRP1 gene.
When placed on the 5xe2x80x2 side of a non-yeast gene the 5xe2x80x2-flanking DNA sequence (promoter) from a yeast gene (for alcohol dehydrogenase 1) can promote the expression of a foreign gene in yeast when placed in a plasmid used to transform yeast. Besides a promoter, proper expression of a non-yeast gene in yeast: requires a second yeast sequence placed at the 3xe2x80x2-end of the non-yeast gene on the plasmid so as to allow for proper transcription termination and polyadenylation in yeast. This promoter can be suitably employed in the present invention-as well as othersxe2x80x94see infra. In the preferred embodiments, the 5xe2x80x2-flanking sequence of the yeast 3-phosphoglycerate kinase gene (37) is placed upstream from the structural gene followed again by DNA containing terminationxe2x80x94polyadenylation signals, for example, the TRP1 (31, 32, 33) gene or the PGK (37) gene.
Because yeast 5xe2x80x2-flanking sequence (in conjunction with 3xe2x80x2 yeast termination DNA) (infra) can function to promote expression of foreign genes in yeast, it seems likely that the 5xe2x80x2-flanking sequences of any highly-expressed yeast gene could be used for the expression of important gene products. Since under some circumstances yeast expressed up to 65 percent of its soluble protein as glycolytic enzymes (38) and since this high level appears to result from the production of high levels of the individual mRNAs (39), it should be possible to use the 5xe2x80x2-flanking sequences of any other glycolytic genes for such expression purposesxe2x80x94e.g., enolase, glyceraldehydexe2x80x943-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose 6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Any of the 3xe2x80x2-flanking sequences of these genes could also be used for proper termination and mRNA polyadenylation in such an expression systemxe2x80x94cf. Supra. Some other highly expressed genes are those for the acid phosphatases (40) and those that express high levels of production due to mutations in the 5xe2x80x2-flanking regions (mutants that increase expression)xe2x80x94usually due to the presence of a TY1 transposable element (41).
All of the genes mentioned above are thought to be transcribed by yeast RNA polymerase II (41). It is possible that the promoters for RNA polymerase I and III which transcribe genes for ribosomal RNA, 5S RNA, and tRNAs (41, 42), may also be useful in such expression constructions.
Finally, many yeast promoters also contain transcriptional control so they may be turned off or on by variation in growth conditions. Some examples of such yeast promoters are the genes that produce the following proteins: Alcohol dehydrogenase II, isocytochrome-c, acid phosphatase, degradative enzymes associated with nitrogen metabolism, glyceraldehydexe2x80x943-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization (39). Such a control region would be very useful in controlling expression of protein productxe2x80x94especially when their production is toxic to yeast. It should also be possible to put the control region of one 5xe2x80x2-flanking sequence with a 5xe2x80x2-flanking sequence containing a promoter from a highly expressed gene. This would result in a hybrid promoter and should be possible since the control region and the promoter appear to be physically distinct DNA sequences.
3. Cell Culture Systems/Cell Culture Vectors
Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years (see 43). The COS-7 line of monkey kidney fibroblasts may be employed as the host for the production of non-human animal interferons (44). However, the experiments detailed here could be performed in any cell line which is capable of the replication and expression of a compatible vector, e.g., WI38, BHK, 3T3, CHO, VERO, and HeLa cell lines. Additionally, what is required of the expression vector is an origin of replication and a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences. While these essential elements of SV40 have been exploited herein, it will be understood that the invention, although described herein in terms of a preferred embodiment, should not be construed as limited to these sequences. For example, the origin of replication of other viral (e.g., Polyoma, Adeno, VSV, BPV, and so forth) vectors could be used, as well as cellular origins of DNA replication which could function in a nonintegrated state.
B. Vector Systems
1. Direct Expression of Mature Bovine Interferon in E. coli 
The procedure used to obtain direct expression of bovine interferon in E. coli as a mature interferon polypeptide (minus signal sequence) involved the combination of a plasmid containing a promoter fragment and translational start signal with a tailored fragment of animal genomic DNA that contained the coding region for the mature interferon.
2. Expression in Yeast
To express a heterologous gene such as the DNA for non-human animal interferon in yeast, it is necessary to construct a plasmid vector containing four components. The first component is the part which allows for transformation of both E. coli and yeast and thus must contain a selectable gene from each organism, such as the gene for ampicillin resistance from E. coli and the gene TRP1 from yeast. This component also requires an origin of replication from both organisms to be maintained as a plasmid DNA in both organisms, such as the E. coli origin from pBR322 and the ars1 origin from chromosome III of yeast.
The second component of the plasmid is a 5xe2x80x2-flanking sequence from a highly expressed yeast gene to promote transcription of a downstream-placed structural gene, such as the 5xe2x80x2-flanking sequence used is that from the yeast 3-phosphoglycerate kinase (PGK) gene.
The third component of the system is a structural gene constructed in such a manner that it contains both an ATG translational start and translational stop signals. The isolation and construction of such a gene is described infra.
The fourth component is a yeast DNA sequence containing the 3xe2x80x2-flanking sequence of a yeast gene, which contains the proper signals for transcription termination and polyadenylation.
3. Expression in Mammalian Cell Culture
The strategy for the synthesis of immune interferon in mammalian cell culture relies on the development of a vector capable of both autonomous replication and expression of a foreign gene under the control of a heterologous transcriptional unit. The replication of this vector in tissue culture can be accomplished by providing a DNA replication origin (derived from SV40 virus), and providing helper function (T antigen) by the introduction of the vector into a cell line endogenously expressing this antigen (46, 47). The late promoter of SV40 virus preceded the structural gene of interferon and ensured the transcription of the gene.
A useful vector to obtain expression consists of pBR322 sequences which provides a selectable marker for selection in E. coli (ampicillin resistance) as well as an E. coli origin of DNA replication. These sequences are derived from the plasmid PML-I (46) and encompasses the region spanning the EcoRI and BamHI restriction sites. The SV40 origin is derived from a 342 base pair PvuII-HindIII fragment encompassing this region (48, 49) (both ends being converted to EcoRI ends). These sequences, in addition to comprising the viral origin of DNA replication, encode the promoter for both the early and late transcriptional unit. The orientation of the SV40 origin region is such that the promoter for the late transcriptional unit is positioned proximal to the gene encoding interferon.